Rat protamine was extracted from S-(pyridylethylated) epididymal sperm cell nuclei with dilute hydrochloric acid. The final purification was achieved by reversed-phase high-performance liquid chromatography. The primary structure was determined by N-terminal sequencing of the total S-(pyridylethylated) protein, and of endoproteinase Lys-C- and thermolysin-derived fragments. Rat protamine consists of 50 amino-acid residues. It is a typical type 1 protamine and differs in two and ten positions from the corresponding mouse and rabbit protamine, respectively. Only 26 positions are invariant in all type 1 mammalian protamines.